The present invention relates generally to novel caspase polynucleotides and polypeptides.
Cysteine-dependent aspartate-specific proteases (caspases) are a family of proteases that cleave their substrates at aspartic acid (D)-X bonds. They are highly specific endopeptidases that catalyze limited proteolysis [Stennicke et al., Cell Death Differen. (1999) 6:1054-1059]. To date 14 mammalian caspases have been identified. Caspase-2, -3, -6, -7, -8, -9 and -10 are major players in the execution phase of apoptosis, whereas caspase-1, -4, -5, and -11 are involved in cytokine processing. The function of caspase-13, and -14 have not been determined, although based on structure caspase-13 is more similar to those caspases active in cytokine processing whereas caspase-14 appears to be closely related to caspases associated with cell death [Slee et al., Cell Death Differen. (1999) 6:1067-1074].
Caspases are initially expressed within cells as zymogens with low or no detectable enzymatic activity. The general primary structure of an unprocessed caspase consists of an amino-terminal prodomain, followed by a large subunit and a small subunit. Proteolytic processing of the zymogen either in trans or cis results in one or more cleavages at specific aspartic acid residues present between the large and small subunits. In most caspases, cleavage also occurs between the prodomain and the large subunit. The mature active caspase is a heterotetramer, which consists of two large (xcx9c20 kDa) and two small (xcx9c10 kDa) subunits. Residues from both subunits contribute to the two substrate-binding pockets which recognize at least four amino acids (P1, P2, P3, and P4) located N-terminal to the cleavage site in substrates. A highly conserved pentapeptide (QACXG) containing the catalytic cysteine, present in the large subunit, leads to the absolute requirement for an Asp at the P1 position. Residues found at positions P2, P3 and P4 on substrates vary greatly and determine substrate specificity within the caspase family [Stennicke et al., Cell Death Differen. (1999) 6:1054-1059; Takahashi, Int J Hematology (1999) 70:226-232; Slee et al., Cell Death Differen. (1999) 6:1067-1074].
Caspases can be divided into two groups based on the length of their N-terminal prodomains. Caspases with long prodomains such as caspase-1, -2, -4, -8, -9, and -10 contain regions such as caspase-recruiting domains (CARD) or death effector domains (DED), that mediate interactions with other proteins. These interactions cause oligomerization of the caspases which is thought to trigger autoprocessing of these proteins. Once activated, these caspases cleave downstream caspases which contain short prodomains and are incapable of autocatalysis, creating a proteolytic cascade [Slee et al., Cell Death Differen. (1999) 6:1067-1074; Kumar, Cell Death Differen. (1999) 6:1060-1066]].
Initial expression as relatively inert zymogens is especially important since most caspases are expressed constitutively. Regulation of caspase activity may also occur by controlling the subcellular localization of caspases. For example, most caspases are present in the cytosol, however, a number of their substrates are localized in organelles such as the nucleus. Catalytically inactive isoforms of caspases, e.g., caspase-9, which are generated by alternative splicing, can interfere with caspase activation thereby inhibiting apoptosis. Some caspases, e.g., caspase-9 can be regulated by post-translational modifications: phosphorylation inhibits its processing and activation. Negative regulators of caspases called inhibitors of apoptosis (IAPs) have also been identified. It is thought that IAPs protect from cell death by interfering with the catalytic activity of caspases or by preventing the processing and activation of caspases [Takahashi, Int J Hematology (1999) 70:226-232; Slee et al., Cell Death Differen. (1999) 6:1067-1074; Kumar, Cell Death Differen. (1999) 6:1060-1066].
Currently, two distinct pathways have been identified that lead to caspase activation in apoptosis: the cell surface death receptor pathway and the mitochondria-initiated pathway. The cell surface death receptor pathway is initiated by ligation of cell surface death receptors, e.g., Fas and tumor necrosis factor receptor 1 (TNFR1), leading to ligand-induced receptor trimerization, recruitment of intracellular receptor-associated proteins as well as caspases with long prodomains, and ultimately to the activation of caspases. The mitochondria-initiated pathway begins with the release of cytochrome c from the mitochondria in response to various stimuli, e.g., DNA damage. Cytosolic cytochrome c binds to apoptotic protease activating factor 1 (Apaf1) forming an Apaf1-cytochrome c multimeric complex, which then associates with caspase-9 thereby triggering the activation of several downstream caspases. Both pathways lead to enzymatically active caspases that cleave substrates including poly (ADP-ribose) polymerase (PARP), PKCxcex4, and cPLA2 [Takahashi, Int J Hematology (1999) 70:226-232; Slee et al., Cell Death Differen. (1999) 6:1067-1074; Kumar, Cell Death Differen. (1999) 6:1060-1066].
Caspases have also been shown to be important for the processing of the cytokines interleukin-1xcex2 (IL-1xcex2) and interleukin-18 (IL-18). Both IL-1xcex2 and IL-18 are expressed as inactive precursor proteins that are proteolytically processed by caspases to yield active cytokines involved in inflammatory responses. IL-1xcex2 is a multifunctional protein present in a number of different cell types. For example, IL-1xcex2 is a growth factor for acute myeloid leukemia cells, is produced by murine monocytes that migrate into Peyer""s patches during inflammation, and is associated with prolonged longevity of peripheral blood monocytes in vitro. IL-18 is a crucial cytokine involved in IFN-xcex3 production in Th1 cells and natural killer cells during inflammation [Zeuner et al., Cell Death Differen. (1999) 6:1075-1080].
Recent studies have also uncovered possible physiological roles for caspases apart from apoptosis. Pro-apoptotic caspase-3 is suggested to be involved in processes such as T cell proliferation, IL-2 release in PHA-stimulated Jurkat T cells, IL-16 processing in CD8+ and CD4+ T cells, and in cell cycle control [Zeuner et al., Cell Death Differen. (1999) 6:1075-1080]. Caspases have also been implicated in regulating terminal lens fiber differentiation. The mature lens fibers do not undergo cell death, however, they do exhibit nuclear degeneration similar to that seem in apoptosis. Finally, caspases have been shown to mediate CD95 inhibition of erythroid differentiation by cleaving specific transcription factors [Zeuner et al., Cell Death Differen. (1999) 6:1075-1080]. Although the most extensively examined process associated with caspase is apoptosis, they appear to function in several other processes.
Of interest to the present invention is Van de Craen et al., FEBS Lett. (1997) 403:61-69, which describes the identification and cloning of murine caspase-12. Murine caspase-12 is predominantly expressed in skeletal muscle and lung, and moderately expressed in brain, heart, spleen, liver, kidney and testis. Northen analysis indicates the presence of several different size murine caspase-12 transcripts suggesting that different isoforms of murine caspase-12 may exist. Transient transfection of murine caspase-12 into HeLa and Rat1 cells resulted in the induction of apoptosis [Van de Craen et al., FEBS Lett. (1997) 403:61-69].
Nakagawa et al., Nature (2000) 403: 98-103, suggests a potential role for mouse caspase-12 in neurodegenerative disease. Nakagawa et al. reported that cortical neurons isolated from caspase-12 null mutant mice were relatively resistant to induction of apoptosis by xcex2-amyloid. Compared to neurons from wild-type animals, neurons from the caspase-12 null mice could be induced to undergo apoptosis by stimuli that trigger cell death pathways which act through the plasma membrane or mitochondria but not through the stress pathways of the endoplasmic reticulum. To date, the human ortholog of mouse caspase-12 has not been identified and isolated.
There thus exists a need in the art for identification and characterization of additional caspases to further elucidate the role of this important family of molecules in pathological conditions and to develop improved treatments for such conditions.
The present invention provides purified polynucleotides encoding heretofore unknown human caspase-12 polypeptides, including species homologs, analogs, and variants especially allelic variants thereof; antisense polynucleotide molecules; constructs and recombinant host cells incorporating the polynucleotides; human caspase-12 polypeptides encoded by the polynucleotides; antibodies to the polypeptides; kits employing the polynucleotides and polypeptides; and methods of making and using all of the foregoing.
The invention is based on cloning of cDNA encoding multiple isoforms of human caspase-12. Partial human caspase-12 amino acid sequences derived from computer-aided analysis of partial human DNA sequences are set forth in SEQ ID NOS: 39, 41, 42, 43, 48 and 49. Nucleotide sequences representing four different human caspase-12 isoforms (designated isoforms A or KW-A, B or KW-B, C or KW-C and D or KW-D) are set forth in SEQ ID NOS: 1 and 3 (KW-A), 5 and 7 (KW-B), 9 (KW-C) and 10 (KW-D). Deduced amino acid sequences for isoforms A and B are set forth in SEQ ID NOS: 2 and 4 (for SEQ ID NOS: 1 and 3, respectively) and 6 and 8 (for SEQ ID NOS: 5 and 7, respectively). An alignment of the amino acid sequence of isoform A (SEQ ID NO: 4), with amino acid sequences of other members of the caspase family is shown in FIG. 1. All four of these human caspase-12 isoforms, and indeed all isoforms known to date, show 60% identity over their entire length to murine caspase-12 at the amino acid level. Predicted cleavage sites within isoform KW-A (SEQ ID NO: 4) based on alignment with other caspases are described below.
Nucleotide sequences of updated versions of KW-A, KW-B, KW-C and KW-D are set forth in SEQ ID NOS: 50, 52, 54 and 56, respectively. The corresponding deduced amino acid sequences are set forth in SEQ ID NOS: 51, 53, 55 and 57, respectively. Nucleotide sequences of additional human caspase-12 isoforms KW-E, KW-F, KW-G, KW-H, KW-I, KW-J, and KW-K are set forth in SEQ ID NOS: 58, 60, 62, 64, 66, 68 and 70, respectively. The corresponding deduced amino acid sequences are set forth in SEQ ID NOS: 59, 61, 63, 65, 67, 69 and 71, respectively. FIG. 2 displays an alignment of all eleven isoforms together with a non-naturally occurring variant containing a Ser-205 to Gly mutation (SEQ ID NO: 77) designated hCaspase-12.
A modified KW-K nucleotide sequence, in which the internal stop codon has been eliminated by changing the T at position 476 to a C and in which a C has been added after position 403 to maintain the open reading frame (designated RIK-2) appears in SEQ ID NO: 72; the deduced amino acid sequence is set forth in SEQ ID NO: 73. A further modification of this nucleotide sequence to delete the CARD region (designated RIK-4) appears in SEQ ID NO: 74; the deduced amino acid sequence is set forth in SEQ ID NO: 75. Yet another modification of RIK-2 to incorporate a Ser205 to Gly mutation by changing the A at position 621 to a G appears in SEQ ID NO: 76; the deduced amino acid sequence is set forth in SEQ ID NO: 77. A further modification of RIK-4 to incorporate the same Ser205 to Gly mutation appears in SEQ ID NO: 78; the deduced amino acid sequence is set forth in SEQ ID NO: 79.
In one embodiment, the invention provides purified and isolated polynucleotides (e.g., cDNA, genomic DNA, synthetic DNA, RNA, or combinations thereof, single or double stranded) that comprise nucleotide sequences encoding the amino acid sequences of the polypeptides of the invention. Such polynucleotides are useful for recombinant expression of protein and also for detecting expression of human caspase-12 in cells (e.g., using Northern hybridization and in situ hybridization assays). Such polynucleotides are also useful to design antisense and other molecules for suppressing the expression of any one of the human caspase-12 polynucleotides or polypeptides of the invention in a cultured cell or animal (for therapeutic purposes or to provide a model for diseases characterized by aberrant expression of any one of the human caspase-12 molecules of the invention). Polynucleotides of the invention are also useful in gene therapy methods. Specifically excluded from the definition of polynucleotides of the invention are entire isolated chromosomes of native host cells.
Exemplary human caspase-12 polynucleotides are set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 10, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78. It will be appreciated that numerous other nucleotide sequences exist that also encode human caspase-12 polypeptides of SEQ ID NOS: 2, 4, 6, 8, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77 or 79 due to the well-known degeneracy of the universal genetic code.
The invention also provides a purified and isolated polynucleotide comprising a nucleotide sequence that encodes a caspase polypeptide, which is preferably capable of biological activity alone or in association with other caspase subunits or domains, wherein the polynucleotide hybridizes to any of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 10, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78; or the non-coding strands complementary to these sequences, under the following exemplary moderately stringent hybridization conditions for human caspase-12:
(a) hybridization for 16 hours at 42xc2x0 C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% Dextran sulphate; and
(b) washing 2 times for 30 minutes at 60xc2x0 C. in a wash solution comprising 0.1% SSC, 1% SDS. Alternatively, highly stringent conditions include washes at 68xc2x0 C. Polynucleotides that encode a human allelic variant are highly preferred.
The invention also provides a purified and isolated polynucleotide comprising a nucleotide sequence that encodes a caspase polypeptide, which is preferably capable of biological activity alone or in association with other caspase subunits or domains, wherein the caspase-encoding portion of the polynucleotide is at least about 99%, at least about 98%, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, or at least about 70% identical over its full length to one of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 10, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 or a subunit- or domain-encoding portion thereof.
Polynucleotides may comprise at least about 100, or at least about 500, contiguous nucleotides of SEQ ID NOS: 1, 3, 5, 7, 9, 10, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78. Preferred polynucleotides of the invention comprise a portion of any one of the sequences set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 10, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 that encodes at least one subunit, domain or truncation of a caspase (e.g. the prodomain, the large subunit and/or the small subunit). The invention also includes polynucleotides differing from the sequences set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 10, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78; and from the complementary strands of these sequences by at least one nucleotide. Other preferred polynucleotides of the invention encode a portion of the amino acid sequence of SEQ ID NOS: 2, 4, 6, 8, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77 or 79 that comprises a subunit, domain or truncation thereof.
Fragment polynucleotides of at least 18 consecutive polynucleotides that are capable of specifically hybridizing to any one of SEQ ID NOS: 1, 3, 5, 7, 9, 10, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 are also contemplated, as are kits comprising such fragments.
Also included within the scope of the invention are polynucleotides encoding human caspase-12 isoforms or variants with amino acid insertions (including fusion proteins), deletions, and/or substitutions, or other allelic variants or splice variants. Specifically contemplated are polynucleotides encoding any one of the amino acid sequences of the polypeptides of the invention fused to a nucleotide sequence encoding a heterologous amino acid sequence.
In a related embodiment, the invention provides DNA constructs comprising any one of the polynucleotides of the invention. Such constructs are useful, e.g., for amplifying the polynucleotides in host cells to create useful quantities thereof. In preferred embodiments, the constructs comprise any one of the polynucleotides of the invention operatively linked to an expression control sequence. Such constructs are useful for recombinant production of polypeptides of the invention.
In another related embodiment, the invention provides host cells that are transformed or transfected (stably or transiently) with polynucleotides of the invention or constructs of the invention or host cells modified to permit or increase expression of endogenous human caspase-12 polynucleotides. Cells can be modified (e.g., by homologous recombination) to provide increased expression of the human caspase-12 polynucleotides of the invention by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cell expresses the human caspase-12 polynucleotides at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to sequences encoding any one of the human caspase-12 polypeptides of the invention. Cells can also be modified to provide increased expression by inserting DNA encoding a heterologous transcription factor that up-regulates human caspase-12 expression. Such host cells are useful for amplifying the polynucleotides and also for expressing any one of the human caspase-12 polypeptide isoforms including a subunit or fragment thereof encoded by the polynucleotide. Such host cells are also useful in assays as described herein.
In another embodiment, the invention provides purified and isolated human caspase-12 polypeptides comprising the amino acid sequence set forth in SEQ ID NOS: 2, 4, 6, 8, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77 or 79; or fragments thereof comprising an epitope specific to any one of the human caspase-12 polypeptides of the invention. By xe2x80x9cepitope specific toxe2x80x9d is meant a portion of any one of the human caspase-12 polypeptides of the invention that is recognizable by an antibody that is specific for one or more of the human caspase-12 polypeptides of the invention, as defined in detail below. An exemplary embodiment is a purified and isolated polypeptide comprising the complete amino acid sequence set forth in SEQ ID NOS: 2, 4, 6, 8, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77 or 79. Also provided are polypeptides comprising a specific subunit, domain or truncation of the polypeptide having an amino acid sequence of SEQ ID NOS: 2, 4, 6, 8, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77 or 79 (e.g. the prodomain, the large subunit and/or the small subunit).
Polypeptides comprising the amino acid sequence set forth in any one of SEQ ID NOS: 39, 41, 42, 43, 48 or 49 are also contemplated.
Although SEQ ID NOS: 2, 4, 6, 8, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77 or 79 provide a particular human sequence, the invention is intended to include within its scope variants with amino acid insertions (including fusion proteins), deletions, and/or substitutions; other human allelic variants, splice variants, or isoforms of the human caspase-12 polypeptides of the invention.
Polypeptides of the invention include polypeptides that are encoded by polynucleotides that hybridize under stringent, preferably highly stringent conditions, to the nucleotide sequences of SEQ ID NOS: 1, 3, 5, 7, 9, 10, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, or the non-coding strand thereof.
Polypeptides of the invention also include polypeptides that are at least about 99%, at least about 98%, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, or at least about 70% identical to one of the amino acid sequences set forth in SEQ ID NOS: 2, 4, 6, 8, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77 or 79 or at least 30 contiguous amino acids thereof.
Polypeptides may comprise at least about 20, or at least about 40, contiguous amino acids of SEQ ID NOS: 2, 4, 6, 8, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77 or 79. In a preferred embodiment, a caspase-12 polypeptide comprises at least one subunit of a human caspase-12. By xe2x80x9csubunitxe2x80x9d is meant a portion of a caspase that is proteolytically cleaved in trans or cis and which portion then associates with the same or different subunits of a caspase to perform an enzymatic activity.
In still another related embodiment, the invention provides a method for producing a human caspase-12 polypeptide (including variants and fragments) comprising the steps of growing a host cell of the invention in a nutrient medium and isolating the polypeptide or variant thereof from the cell or the medium.
In still another embodiment, the invention provides antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR and/or antigen-binding sequences, which specifically recognize a polypeptide of the invention) and other binding proteins specific for any one of the human caspase-12 polypeptides of the invention. Antibodies are useful for detecting or quantitating human caspase-12 polypeptides or for inhibiting the activity of human caspase-12. Antibody specificity and cross-reactivity is described in greater detail below. The determination of whether an antibody is specific for any one of the human caspase-12 polypeptides of the invention or is cross-reactive with another known caspase may be made using Western blotting assays or several other assays well known in the literature.
In one preferred variation, the invention provides monoclonal antibodies. Hybridomas that produce such antibodies are also intended as aspects of the invention. In yet another variation, the invention provides a humanized antibody. Humanized antibodies are useful for in vivo therapeutic applications.
In another variation, the invention provides a cell-free composition comprising polyclonal antibodies, wherein at least one of the antibodies is an antibody of the invention specific for one or more of the human caspase-12 isoforms. Antisera isolated from an animal is an exemplary composition, as is a composition comprising an antibody fraction of an antisera that has been resuspended in water or in another diluent, excipient, or carrier.
In still another related embodiment, the invention provides an anti-idiotypic antibody specific for an antibody that is specific for one or more of the human caspase-12 polypeptide isoforms.
Also within the scope of the invention are compositions comprising polypeptides, polynucleotides, antibodies, or other modulators of the invention that have been formulated with, e.g., a pharmaceutically acceptable carrier.
The invention also provides assays to identify compounds that bind to and/or inhibit human caspase-12. One such assay comprises the steps of: (a) contacting a composition comprising a human caspase-12 polypeptide with a test compound; and (b) measuring binding between the compound and human caspase-12. The binding may be measured by any one of numerous methods known in the art. Exemplary assay formats are described herein.
The invention also provides a method for identifying a modulator (e.g., inhibitor or enhancer/activator) of human caspase-12 enzymatic activity comprising the steps of: (a) contacting a human caspase-12 substrate and a composition comprising a human caspase-12 polypeptide in the presence and in the absence of a putative modulator compound; (b) detecting proteolytic (enzymatic) activity of the human caspase-12 substrate; and (c) identifying a putative modulator compound in view of decreased or increased proteolytic activity of human caspase-12 in the presence of the putative modulator, as compared to proteolysis in the absence of the putative modulator. Also provided are methods to identify modulators of binding of human caspase-12 to adaptor molecules, receptor molecules, substrates or other ligands, e.g., comprising the steps of (a) contacting a human caspase-12 polypeptide with a binding partner in the presence and absence of a test compound, and (b) detecting binding of the caspase polypeptide to the binding partner, wherein a decrease in binding indicates that the test compound is an inhibitor of binding.
Further, the invention provides a method for identifying a candidate activator of human caspase-12 comprising the steps of contacting a composition comprising a caspase polypeptide lacking an active site sequence, a caspase polypeptide having an active site sequence and a substrate, and measuring enzymatic activity of said composition in the presence and absence of a test compound, wherein a change in enzymatic activity means that the test compound is a candidate modulator.
A number of the caspases identified have been characterized as important effector molecules for apoptosis, whereas other caspases are involved in cytokine processing associated with inflammation. The invention provides a method for treating a disease or disorder associated with inappropriate apoptosis or abnormal inflammation (including any diseases or disorders described in further detail herein) caused by activation of human caspase-12 comprising the step of administering to a mammal in need of such treatment an amount of a caspase inhibitor of the invention (e.g., antibodies, antisense polynucleotides, or other inhibitors, including inhibitors identified by the screening methods of the invention) that is sufficient to inhibit activation of human caspase-12 (i.e., a therapeutically effective amount), thereby inhibiting apoptosis or inhibiting cytokine processing. The invention also provides a method of inducing apoptosis by administering human caspase-12 polynucleotides or polypeptides or agonists of the invention for therapeutic use as anti-viral or anti-tumor agents. Treatment methods using small molecules that mimic, agonize or antagonize the activation of human caspase-12, including small molecules identified by the screening methods of the invention, are also contemplated. Treatment of individuals having any of these disorders is contemplated as an aspect of the invention.
Use of any of the human caspase-12 polynucleotides, polypeptides, inhibitors or agonists of the invention in preparation of a medicament for the treatment of any of the disorders described herein is also contemplated. Thus, the invention provides a method of using one or more of these products, such as a compound that binds to and/or inhibits human caspase-12, in the manufacture of a medicament for preventing or treating a disorder involving, e.g., inappropriate apoptosis and/or excessive cell proliferation, such as an inflammatory disease, a neurodegenerative disease, cancer, a cardiovascular disease and, indeed, any disorder or disease characterized by a gradual and prolonged development of apoptosis.
Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.
In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. Although the applicant(s) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention.